INTRODUCTION:

Tuberculosis is an infectious disease that causes death that can be cured. Based on the results of surveys of the prevalence of infection and disease, an assessment of the effectiveness of surveillance systems, and death registries, it is estimated that there were 8.9 million new

cases of tuberculosis in 2004, less than half of which were reported to public health authorities and WHO. About 3.9 million are smear positive cases, the most contagious form of the disease. The majority of patients with tuberculosis live in Asia's most populous country; such as Bangladesh, China, India, Indonesia, and Pakistan contributed half (48%) of new cases appear each year. About 80% of people newly diagnosed with this disease each year live in the 22 most populous countries¹^,2,3.

The development of a TB diagnosis is the initial step and determines the next series of processes, namely treatment, the success of a treatment will not be achieved without being based on a precise and accurate diagnosis. The definite diagnosis of tuberculosis includes the presence of M. tuberculosis through microbiological smear staining, this method has limitations for the diagnosis of extrapulmonary tuberculosis. The culture method takes a long time of 6-8 weeks, whereas patients often need a prompt diagnosis. Materials for the diagnosis of PCR are often impossible to take (except spinal fluid and cerebrospinal fluid), and tissue biopsy for histopathological examination is an invasive measure.

TB diagnostic techniques other than standard techniques are needed, such as the detection of new biochemical markers to diagnose this infection. In this study we examined serum adenosine deaminase levels as a diagnostic parameter for Extra Pulmonary Tuberculosis (EPTB).

In previous studies, the importance of serum adenosine deaminase levels as a parameter can be used in diagnosing extrapulmonary tuberculosis, but its significance in making the diagnosis still requires further clinical research⁴.

In the diagnosis of tuberculosis (TB) is still a problem in clinical practice. Conventional diagnostic methods are very useful but lack sufficient sensitivity and specificity. Adenosine deaminase (ADA) enzyme assay has been developed and is widely used for the diagnosis of TB. ADA is an enzyme that is elevated in Tuberculosis due to stimulation of mycobacterium antigens to T-lymphocytes. There are sufficient data to support the results of ADA in various body fluids for the diagnosis of TB. ADA can be used for early diagnosis of tuberculosis, especially in cases of negative acid-resistant bacilli (BTA) smears^5,6,7.

MATERIAL AND METHODS:

This research is an analytic observational study with a cross sectional study method. This research was conducted for 8 months (March 2020 - November 2020) with the activities of submitting ethical tests, taking blood samples from tuberculosis patients and examining ADA serum using the ELISA method at Dr Ramelan Hospital Surabaya Indonesia, and conducted standard PCR method on gyrB region gene in the Institute of Tropical Disease, Airlangga University, and data analysis.

The sample size used was total sampling, which is the total number of patients diagnosed with Pulmonary Tuberculosis and Extrapulmonary Tuberculosis in the Lung Poli Dr Ramelan Hospital Surabaya for 8 months (January-September 2020).

Diagnosis of Pulmonary Tuberculosis and Extrapulmonary Tuberculosis is enforced by fellow pulmonary clinicians on duty. Where as the experimental unit was blood serum in each group diagnosed with pulmonary tuberculosis and extrapulmonary tuberculosis. In each group, 6 cc of venous blood was then checked for Adenosine Deaminase levels at the Tuberculosis Laboratory of the Tropical Disease Institute, Airlangga University, Surabaya, Indonesia.

RESULTS AND DISCUSSION:

It was found that new patients diagnosed with TB at RSAL Dr. Ramelan had the following descriptive data.

Table 1: Frequency of TB patients based on genders

Valid	Gender	Frequency	Percent
	Male	101	57.4
	Female	75	42.6
	Total	176	100.0

Figure 1: Gender distribution of RSAL Dr. Ramelan TB patients

In terms of gender, a larger proportion is male, the number of male sufferers is mostly found in tuberculosis patients at RSAL Dr. Ramelan. This is related to the smoking habit in men, which makes their scar tissue relatively sensitive to the inflammatory process in Mycobacterium tuberculosis infection. Another factor is that more men work outside the home, allowing greater exposure to infection.

Based on the age distribution of the sample, it was found that the most age was found in the age group over 50 years old, the risk of inflammation would increase with age. The second largest group is the 30-40 years age group which is the productive age group which allows this age group to be outside the risk of exposure to tuberculosis.

Meanwhile, the smallest group is in the age group less than 20 years, where this age group still has a relatively good immune response. Also in this group an average age-limited student group accepts the risk of exposure to infection and inflammation from the learning environment.

As many as 176 samples, the largest number was in the pulmonary TB group as many as 122, while the extrapulmonary TB group which included TB spondylitis, glandular TB, TB meningitis and so on were 40 people. Meanwhile, the group with features of pulmonary and extra-pulmonary TB was relatively small, there are 8 people.

It can be seen that most of the TB patients at RSAL Dr. Ramelan came to the hospital with a main complaint of prolonged cough, or were referred from satellite puskesmas with a suspected diagnosis of pulmonary tuberculosis.

Table 2: Number of TB patients with pulmonary and extrapulmonary symptoms

Valid	TB	Frequency	Percent
	Pulmonal	122	69.3
	Extrapulmonal	44	25.0
	Pulmonal and extrapulmonal	10	5.7
	Total	176	100.0

Figure 2: Distribution of pulmonary TB patients and extrapulmonary TB patients

From the levels of adanosin deaminase, the levels were the same in the three types of tuberculosis, so there was no difference between the three groups.

Table 3: The level of adenosine deaminase (U/L) of TB patients

Tuberculosis	Adenosine deaminase (U/L)
Pulmonal TB	20.05
Extrapulmonal TB	17.14
Pulmonal & extrapulmonal TB	20.23

Figure 3: Distribution of adenosine deaminase (U/L) level of TB patients

The positive AFB is only a little and less than half of the sample size. The largest number was BTA negative TB, as many as 122 people. Diagnosis of tuberculosis in patients with smear negative is based on chest X-ray images and clinical symptoms.

Table 4: Data of BTA frequency of TB patients

Valid	BTA	Frequency	Percent
	Negative	139	79.0
	< 1	14	8.0
	1-10	3	7.4
	>10	10	5.7
	Total	176	100.0

Figure 4: Proportion of the number of BTA.

Furthermore, the Kolmogorov smirnov normality test was carried out on the three groups of tuberculosis types, namely the pulmonary, extrapulmonary and pulmonary and extrapulmonary tuberculosis group. It appears that one group shows a significance of 0.000 (<0.005) meaning that the distribution is not normal.

Because the distribution is not normal, the test performed is a non-parametric statistic. The Kruskall Wallis test was used here to see the differences in the three groups. The results of statistical tests showed the value of p = 0.116, where the value was> 0.005 (p> α), meaning that there was no significant difference in the three groups of tuberculosis types, namely the pulmonary, extrapulmonary and combined pulmonary and extrapulmonary groups.

Furthermore, the Kruskal Wallis test was carried out to see the difference in the mean levels of adenosine de aminase based on the BTA category, a significance level was obtained with a p value of 0.011 (p <α) which means that there is a significant difference in the mean levels of adenosine de aminase to the BTA category.

Furthermore, the Mann Whitney U test was performed to determine which mean ADA was significant in which group. The results showed that the mean ADA was a significant difference between group 0 (negative AFB) and group 3 (positive AFB 3) (p value = 0.002), also between group 1 (positive AFB 1) and group 3 (positive AFB 3) (value p = 0.012) and the last is between the BTA positive 2 and the positive 3 BTA (p value = 0.012).

After that, the specificity test, negative sensitivity predictive value and positive predictive value were carried out on the results of the adenosine de aminase enzyme level examination. In medical studies, diagnostic tests are used to determine the presence or absence of disease in the observed subject. In this case, to test the presence or absence of tuberculosis infection through examination of ADA levels. The diagnostic test for the examination of ADA levels was validated by comparing the test results with the gold standard of the diagnosis of tuberculosis, in this case the BTA examination. Validation of this test is used to determine and assess how good the test is in making a diagnosis. Validation involves calculating four objective measures of test performance, namely, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Ideally, a diagnostic test will correctly identify subjects with and without disease with 100% accuracy^8,9.

AUC (Area Under Curve) shows how well this ROC curve model is for predicting BTA. The AUC value ranges from 0-1, the closer to 1, the better the ROC model. In this study, AUC = 0.473 = 47.30%.

Analysis of the results of the ROC (Receiver Operating Characteristic) curve shows high sensitivity and specificity when the ROC curve approaches the upper left corner of the box. The ROC curve is used to differentiate the diagnosis of Tuberculosis based on the BTA (Acid Resistant Rod) examination and based on the examination of the ADA concentration. The results of the ROC in this study were near the 45 degree diagonal line, meaning that the ADA concentration had low sensitivity and sensitivity to predict the presence of BTA.

This means that the strength of the ADA level to be used as a differentiator in the diagnosis of tuberculosis with BTA examination is not good (low). ADA levels are used as a screening tool to differentiate the diagnosis of tuberculosis from non-tuberculosis based on BTA examination.

The examination of the adenosine deaminase enzyme in the pleural fluid of tuberculous pleural effusion patients at the Dr. Soetomo Surabaya Hospital, it was stated that this enzyme is of diagnostic value in diagnosing pleural effusion caused by tuberculosis. In this study, 18 samples with tuberculous pleural effusions and 30 samples of non-tuberculous pleural effusions showed significant results between adenosine deaminase levels between the tuberculosis and non tuberculosis groups. However, there were no significant results on the microscopic examination of BTA and the results of the culture^10,11,12.

The same results were also obtained in Rosfadilla's study, namely that there were significant ADA levels in the tuberculosis pleural effusion group with non tuberculosis pleural effusions in a sample of patients at RSUP H Adam Malik Medan⁶.

However, our study did not show the same results as the two studies above, where there were no significant results of adenosine deaminase levels between the pulmonary and extra pulmonary tuberculosis groups, nor significant results in the smear positive and negative smear groups.

200 subjects diagnosed with tuberculosis were divided into 2 groups, namely tuberculosis with a negative smear result and a group with positive smear results. Where this study shows that measurement of serum Adenosine Deaminase levels does not have sufficient sensitivity to assist in the diagnosis of tuberculosis patients with the diagnosis of other respiratory tract infections, nor is it good enough to replace a smear sputum microscope (AFB examination). It was concluded that in this study, the adenosine deaminase enzyme examination was said to have a small role in diagnosing pulmonary tuberculosis¹¹.

TB pleural effusion is the most common extrapulmonary TB after TB lymphadenitis. Limited diagnostic methods make TB pleural effusion difficult to diagnose. Adenosine deaminase ADA is an enzyme in purine catabolism that catalyzes adenosine to inosine and deoxyadenosine to deoxyinosin. This process is important in lymphoid cell differentiation. ADA is increased in TB pleural effusion⁵.

The ADA level of 100 serum of newborns vaccinated with BCG showed a significant increase, suggesting a human cell-mediated immune response to mycobacterium antigen. Serum ADA levels of 51 children with confirmed tuberculosis (pulmonary, peritoneal, meningeal, and skeletal), and 20 healthy controls showed significant improvement in the first group with p value <0.001. Collazos et al performed a prospective follow-up study of 25 cases of pulmonary and pleural tuberculosis with normal immune responses for a period of 6 months after starting treatment. There was a significant reduction in serum ADA values during the first two months of the patient as a whole (p = 0.04), followed by stabilization of serum ADA activity. This decrease was caused by decreased serum ADA activity in which 13 patients (52%) had high baseline enzyme levels (p = 0.03), whereas patients with normal baseline levels did not change (p = 0.27). Similar results were obtained by Ishii et al. in Japan along with a direct relationship between serum ADA levels and erythrocyte sedimentation rate. Joshaghani et al. demonstrated that the assessment of this enzyme in serum can be a useful method of differentiating healthy subjects from respiratory diseases, but this test lacks sufficient sensitivity to aid in diagnosing tuberculosis patients from other respiratory diseases^13,14,15.

Conde et al evaluated serum ADA in active pulmonary tuberculosis and other pulmonary infections and showed no significant difference between the two. The results are not consistent with the report of Yasuhara et al. in which serum ADA activity of children with active pulmonary tuberculosis was found to be significantly greater than those with bacterial or viral pneumonia. ADA serum activity has good specificity and a positive predictive value. For several years, the ability to differentiate between pulmonary tuberculosis and non-pulmonary tuberculosis diagnoses has been an important issue for clinicians. Examination of serum ADA levels is one method with varying results⁷.

High ADA levels are not only in tuberculosis patients. Several other diseases also produce ADA levels, namely cases of malignant pleural effusion (malignancy). In general, malignant pleural effusions have lower levels of ADA than those found in TB. However, pleural effusions secondary to lymphoma and leukemia which are hematologic malignancies have higher levels of ADA compared to non-hematologic malignancies. This causes haematological malignancies to be difficult to distinguish from tuberculosis. Some of the other diseases that increase ADA levels are rheumatoid arthritis, sarcoidosis, HIV-AIDS and psoriasis. Meanwhile, those that reduce ADA levels are thymic cell death, adenosine deaminase gene mutations and consumption of coformycin drugs^{16,17,18,19,20}.

This study still has several limitations, including not all diagnosis of tuberculosis can be done with the gold standard, namely the discovery of AFB in sputum or pleural fluid or by histopathological examination of pleural tissue biopsy. Most of the TB cases in this study were diagnosed clinically based on the results of the history and physical examination as well as supporting radiological images. This allows for a sampling bias in the study, namely the error of including a subject in the true tuberculosis group and a subject who is not tuberculosis. Therefore, in the future, a better diagnostic test using gold standards such as pleural histopathology biopsy or PCR examination is needed to confirm cases of tuberculosis.

CONCLUSION:

Examination of adenosine deaminase enzyme levels in diagnosing tuberculosis has a low sensitivity, high specificity in tuberculosis patients. This means that it is indicated that this test does not have sufficient sensitivity to assist in the diagnosis of tuberculosis patients compared to other respiratory diseases and is not evaluated as being sufficiently good to replace microscopic examination of sputum smear (AFB). In this study, there were no significant results between Adenosine Deaminase (ADA) serum in diagnosing Pulmonary Tuberculosis and Extra Pulmonary Tuberculosis at Dr. Ramelan Hospital Surabaya, Indonesia. So in this study, this test had a minor role in the diagnosis of pulmonary tuberculosis. From this study we conclude that serum ADA activity is not a useful test for differentiating tuberculosis from other respiratory diseases, and can only be an additional test especially if the diagnosis of tuberculosis is in doubt.

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Received on 24.07.2021 Modified on 30.08.2021

Research J. Pharm. and Tech. 2022; 15(7):2987-2991.

DOI: 10.52711/0974-360X.2022.00498